PLC-γ1 Signaling Pathway and Villin Activation Are Involved In Actin Cytoskeleton Reorganization Induced by Na+/Pi Cotransport Up-regulation

Abstract

IntroductionThe actin cytoskeleton participates in several cellular processes, including intracellular signaling, membrane trafficking, secretion, exoand endocytosis (1)(2)(3)(4)(5). Interactions between the actin cytoskeleton and the plasma membrane are thought to be important in many cellular events, including transport protein activity (6-9). The actin cytoskeleton may affect transport activity directly, by connecting transport proteins subunits to cytoskeletal elements Abstract Background: The brief incubation of opossum kidney (OK) cells with low P i results in Na ϩ /P i cotransport up-regulation and in substantial, but transient, cytoskeletal reorganization. In this study, we examined signaling events involved in the depolymerization of microfilaments. Results: Confocal laser scanning microscopy, immunoblot and immunoprecipitation experiments revealed villin co-localization with mainly actin short filaments and monomers, indicating that under the conditions used, villin acted as an actin-severing protein. Further analysis revealed that low concentrations of extracellular phosphate resulted in phospholipase CͲ1 (PLC-Ͳ1) translocation to the actin cytoskeleton, without increases in its tyrosine phosphorylation. Additionally, tyrosine phosphorylation

Interaction of Phospholipase C-γ1with Villin Regulates Epithelial Cell Migration

“…These data are consistent with our previous reports demonstrating the association of tyrosine-phosphorylated villin with PLC-␥ 1 (1,18). 2 In cells expressing VIL/ANFM, PLC-␥ 1 formed a complex with the mutant villin protein, indicating that amino-terminal phosphorylation sites are not involved in PLC-␥ 1 binding. In contrast, in cells expressing either VIL/ACFM or VIL/AYFM, PLC-␥ 1 did not form a complex with villin.…”

“…More recent studies have established that examining epithelial cell migration in the context of proteins that are uniquely expressed in epithelial cells, including the actin-binding protein, villin is important to our understanding of this rather complex process (14). 2 We have demonstrated that overexpression of villin enhances cell migration (19). Likewise Athman et al (14) have demonstrated that villin enhances hepatocyte growth factor (HGF)-induced actin remodeling in epithelial cells.…”

“…Furthermore, we know from previous studies that PLC-␥ 1 is required for villin-induced increase in cell migration. 2 Based on these previous studies, we hypothesized that the COOH-terminal phosphorylation sites in villin may determine the association of villin with PLC-␥ 1 . To test this hypothesis, we examined the coimmunoprecipitation of villin with PLC-␥ 1 in MDCK Tet-Off cells ectopically expressing VIL/FL, VIL/ACFM, VIL/ANFM, or VIL/AYFM.…”

“…We have demonstrated previously that villin is tyrosine-phosphorylated in vitro and in vivo by c-Src kinase (18,19). 2 Likewise, gelsolin, fragmin, and CapG have been shown to be tyrosine-phosphorylated in vitro by c-Src (20). Furthermore, proteomic analysis of phosphotyrosyl proteins in human lumbar cerebrospinal fluid has been shown to include tyrosine-phosphorylated gelsolin (21).…”

“…In intestinal epithelial cells as well as in the opossum kidney cell line, tyrosine phosphorylation of villin regulates ion transport (1,2). In addition, rearrangement of the microvillar cytoskeleton and concomitant redistribution of villin have been reported in studies with enteroinvasive bacteria (3), in intestinal restitution (4,5), in renal ischemia, in the ischemia of the small intestine (6 -9), and in colonic adenocarcinomas (10).…”